CN104422677A - Analysis method of mushrooms - Google Patents
Analysis method of mushrooms Download PDFInfo
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- CN104422677A CN104422677A CN201310407412.2A CN201310407412A CN104422677A CN 104422677 A CN104422677 A CN 104422677A CN 201310407412 A CN201310407412 A CN 201310407412A CN 104422677 A CN104422677 A CN 104422677A
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Abstract
The invention relates to an analysis method of mushrooms. The method can be used for rapidly measuring effective components of the mushrooms qualitatively and quantitatively; a three-dimensional fluorescence excitation emission test system is used for analyzing mushroom samples; the excitation wavelength and the emission wavelength of the three-dimensional fluorescence test system are respectively set to be 250nm and 310nm.
Description
Technical field
The invention relates to a kind of mushroom Mushrooms analytical approach, particularly about a kind of can the analytical approach of effective constituent of Rapid Screening mushroom Mushrooms.
Background technology
Mushroom Mushrooms are rich in polysaccharide body, can stimulating expression of macrophage activation, secreting tumor necrosis factor (TNF) and white blood cell Jie element-2(IL-2), enhancing antibody produces, and thus has good anti-cancer effect.Wherein, special in Antrodia camphorata, except polysaccharide is external, separately have up to more than 200 kinds of triterpenes components, there is inhibition tumor cell growth, repair liver and promote liver function, reduce blood fat and the function such as blood pressure, lifting immunocompetence, be thus considered to the splendid holy product of health care.
Because mushroom Mushrooms are considered to have splendid tumor suppression curative effect, many dealers research and develop various mushroom Mushrooms and cultivate or extracting process, to promote the active constituent content of mushroom Mushrooms extract; Just, existing mushroom Mushrooms analytical approach but has following defect:
Existing mushroom Mushrooms analytical approach, be DNA (deoxyribonucleic acid) (the deoxyribonucleic acid of extraction one mushroom Mushrooms sample, be called for short DNA), with a polymerase chain reaction (polymerasechain reaction, be called for short PCR) or analyze with an instant polymerase chain reaction (real-time chainreaction is called for short real-time PCR); But this existing mushroom Mushrooms analytical approach only in order to analyze the kind of this mushroom Mushrooms sample, can cannot carry out qualitative or quantitative test to the effective constituent contained by this mushroom Mushrooms sample.
Another existing mushroom Mushrooms analytical approach, then with reverse-phase chromatography method (reversed phasechromatography, as used C18 chromatography tubing string) mushroom Mushrooms sample is analyzed, although can main compound (i.e. the effective constituent of this mushroom Mushrooms sample) that is qualitative and quantitatively this mushroom Mushrooms sample, but, the time that this reverse-phase chromatography method is not only analyzed is longer, after each sample analysis, the extra time is needed again to rinse chromatography tubing string, and need reply after equilibrium state until this chromatography tubing string, just can carry out the analysis of next testing sample, quite expend time in, therefore large-scale testing sample screening is not suitable for.
Accordingly, necessary provides a kind of mushroom Mushrooms analytical approach, to solve the problem.
Summary of the invention
Fundamental purpose of the present invention is to provide a kind of mushroom Mushrooms analytical approach, with the effective constituent of qualitative and quantitative mushroom Mushrooms sample, and promotes the quality of commercially available mushroom Mushrooms commodity.
Another object of the present invention is to provide a kind of mushroom Mushrooms analytical approach, with the effective constituent of quick test mushroom Mushrooms sample, and saves analysis time.
For reaching aforementioned invention object, the technological means that the present invention uses and including by effect that this technological means can reach:
Mushroom Mushrooms analytical approach of the present invention, comprises: step (a): provide a mushroom Mushrooms sample; Step (b): analyze this mushroom Mushrooms sample with a three-dimensional fluorescence detection system, excitation wavelength and the emission wavelength of this three-dimensional fluorescence detection system are respectively 250nm and 310nm.
Mushroom Mushrooms analytical approach of the present invention, wherein, better before analyzing this mushroom Mushrooms sample with this three-dimensional fluorescence detection system, the mushroom Mushrooms sample of a purifying isolated by this mushroom Mushrooms sample step (a) provided through molecular screening tomographic system, the continuous mushroom Mushrooms sample analyzing this purifying with this three-dimensional fluorescence detection system.
Mushroom Mushrooms analytical approach of the present invention, wherein, the molecular weight of the mushroom Mushrooms sample of this purifying is 100 ~ 10000Da.
Mushroom Mushrooms analytical approach of the present invention, wherein, this mushroom Mushrooms sample in this step (a) is preferably mushroom Mushrooms extract through being diluted to clear, and filters with 0.22 μm of filtering membrane.
Mushroom Mushrooms analytical approach of the present invention is the molecular characterization by detecting mushroom Mushrooms extract, can effective constituent that is qualitative and quantitatively this mushroom Mushrooms extract, to reach the effect promoting commercially available mushroom Mushrooms extract quality.
Mushroom Mushrooms analytical approach of the present invention, the excitation wavelength and emission wavelength that have by the molecular structure of mushroom Mushrooms extract, to calculate the active constituent content of this mushroom Mushrooms extract, significantly can reduce the time detected and analyze, reach effect of quick testing sample screening.
Accompanying drawing explanation
Fig. 1 is the process flow diagram of the mushroom Mushrooms analytical approach of the present invention first preferred embodiment.
Fig. 2 A is the outside drawing of the Antrodia camphorata dilution of the 1st table.
Fig. 2 B is the outside drawing of the Antrodia camphorata filtered fluid of the 1st table.
Fig. 3 is the Antrodia camphorata dilution of the 1st table and the granularmetric analysis result figure of Antrodia camphorata filtered fluid.
Fig. 4 A is the three-dimensional fluorescence detection system analysis result figure of A1-1 group Antrodia camphorata dilution through the present invention first preferred embodiment.
Fig. 4 B is the three-dimensional fluorescence detection system analysis result figure of A2-1 group Antrodia camphorata dilution through the present invention first preferred embodiment.
Fig. 4 C is the three-dimensional fluorescence detection system analysis result figure of A3-1 group Antrodia camphorata dilution through the present invention first preferred embodiment.
Fig. 4 D is the three-dimensional fluorescence detection system analysis result figure of A4-1 group Antrodia camphorata dilution through the present invention first preferred embodiment.
Fig. 5 A is the three-dimensional fluorescence detection system analysis result figure of A1-2 group Antrodia camphorata filtered fluid through the present invention first preferred embodiment.
Fig. 5 B is the three-dimensional fluorescence detection system analysis result figure of A2-2 group Antrodia camphorata filtered fluid through the present invention first preferred embodiment.
Fig. 5 C is the three-dimensional fluorescence detection system analysis result figure of A3-2 group Antrodia camphorata filtered fluid through the present invention first preferred embodiment.
Fig. 5 D is the three-dimensional fluorescence detection system analysis result figure of A4-2 group Antrodia camphorata filtered fluid through the present invention first preferred embodiment.
Fig. 6 is the process flow diagram of the mushroom Mushrooms analytical approach of the present invention second preferred embodiment.
Fig. 7 is the rating curve figure that the molecular screening tomographic system of the present invention second preferred embodiment is drawn with molecular weight standards and acetone.
Fig. 8 is the molecular screening tomographic system analysis result figure of A4-2 group Antrodia camphorata filtered fluid through the present invention second preferred embodiment.
Fig. 9 is the three-dimensional fluorescence detection system analysis result figure of A4-2 group Antrodia camphorata filtered fluid through the present invention second preferred embodiment.
Figure 10 A is the molecular screening tomographic system analysis result figure of Antrodia camphorata index components (4,7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene) through the present invention second preferred embodiment.
Figure 10 B is the molecular screening tomographic system analysis result figure of Antrodia camphorata index components (horse is together with No. 1) through the present invention second preferred embodiment.
Figure 11 A is the three-dimensional fluorescence detection system analysis result figure of Antrodia camphorata index components (4,7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene) through the present invention second preferred embodiment.
Figure 11 B is the three-dimensional fluorescence detection system analysis result figure of Antrodia camphorata index components (horse is together with No. 1) through the present invention second preferred embodiment.
Figure 12 A is that the Antrodia camphorata index components (4,7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene) of variable concentrations is in wavelength 205nm absorption value graph of a relation.
Figure 12 B is that Antrodia camphorata index components (4,7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene) is in wavelength 380nm absorption value graph of a relation.
Figure 12 C is that the Antrodia camphorata index components (horse is together with No. 1) of variable concentrations is in wavelength 205nm absorption value graph of a relation.
Figure 12 D is that the Antrodia camphorata index components (horse is together with No. 1) of variable concentrations is in wavelength 380nm absorption value graph of a relation.
Figure 13 is that the Antrodia camphorata index components (horse is together with No. 1) of variable concentrations is in wavelength 270nm absorption value graph of a relation.
Figure 14 is the three-dimensional fluorescence detection system analysis result figure of Cordyceps sinensis index components (cordycepin) through the present invention second preferred embodiment.
Figure 15 is the three-dimensional fluorescence detection system analysis result figure of Cordyceps sinensis index components (cordycepin) through the present invention second preferred embodiment.
Embodiment
For above-mentioned and other object of the present invention, feature and advantage can be become apparent, preferred embodiment of the present invention cited below particularly, and coordinate appended accompanying drawing, be described in detail below:
Please refer to shown in Fig. 1, be the first preferred embodiment of the present invention, the mushroom Mushrooms analytical approach of this first preferred embodiment comprises: provide a mushroom Mushrooms sample, and analyzes this mushroom Mushrooms sample with a three-dimensional fluorescence detection system.
In details of the words, mushroom Mushrooms sample can be extracted by a solvent, and this solvent can be water or various organic solvent, make this effective constituent (as polysaccharide body or triterpenes) contained by mushroom Mushrooms can stripping in this solvent, or, these mushroom Mushrooms also can extract with supercritical carbon dioxide, are not limited at this.
Preferably, mushroom Mushrooms sample can dilute with solvent (namely in order to carry out the solvent extracted), this mushroom Mushrooms sample is made to be clear, and filter with 0.22 μm of filtering membrane (aperture), assemble to avoid the polysaccharide body in the colourity of this mushroom Mushrooms sample and this mushroom Mushrooms sample the sentence read result affecting subsequent analysis.
Then, continue and above-mentioned mushroom Mushrooms sample is analyzed with three-dimensional fluorescence detection system, its initial and final excitation wavelength is set as 200 ~ 900nm, initial and final emission wavelength is set as 200 ~ 900nm, to excite and emission wavelength grating is 10nm, scanning is set as 30000nm/min, and, select the photomultiplier into 700V; Preferably, the excitation wavelength of this three-dimensional fluorescence detection system and emission wavelength are respectively 250nm and 310nm.
For confirming that the mushroom Mushrooms analytical approach of the first preferred embodiment can effective constituent contained by effectively qualitative and quantitative mushroom Mushrooms sample, getting an Antrodia camphorata extract and testing; Wherein, this Antrodia camphorata extract is through obtaining with under type: get an Antrodia camphorata dried powder, extract with an alcohol solvent (concentration is for 95%), the ratio of this Antrodia camphorata dried powder and this alcohol solvent is 1:1(w:v), after re-extract three times, to obtain an Antrodia camphorata stoste, and this Antrodia camphorata stoste is concentrated, to obtain this Antrodia camphorata extract.
Please refer to shown in the 1st table, above-mentioned Antrodia camphorata extract carries out dilution 10 × (A1-1 group) respectively with alcohol solvent (concentration is for 95%), 100 × (A2-1 group), 1, 000 × (A3-1 group) and 10, 000 × (A4-1 group), to obtain the Antrodia camphorata dilution of each group, and the cumulative volume of quantitatively each group Antrodia camphorata dilution is 50mL, the each group of Antrodia camphorata dilution getting 15mL is respectively inserted in centrifuge tube and is preserved, all the other samples are then with 0.22 of ethanol-tolerant μm of acetate fiber filter membrane (Cellulose acetate ester, purchased from Advantec MFS Inc., USA) filter, to obtain the Antrodia camphorata filtered fluid (respectively called after A1-2 ~ A4-2 group) of each group.
The extension rate that 1st shows, this tests each group of Antrodia camphorata dilution and Antrodia camphorata filtered fluid
| Extension rate (×) | Antrodia camphorata dilution | Antrodia camphorata filtered fluid |
| 10× | A1-1 | A1-2 |
| 100× | A2-1 | A2-2 |
| 1,000× | A3-1 | A3-2 |
| 10,000× | A4-1 | A4-2 |
Please refer to shown in Fig. 2 A, obvious sediment thing is there is in the Antrodia camphorata dilution of A1-1 group after leaving standstill, and, along with extension rate increases, the color of each group Antrodia camphorata dilution changes from water white transparency (A4-1 group) from coffee-like (A1-1 group), yellow (A2-1 group), light yellow (A3-1 group).Continuous with reference to shown in Fig. 2 B, also obvious sediment thing is there is in the Antrodia camphorata filtered fluid of A1-2 group after leaving standstill, further, the color of each group Antrodia camphorata filtered fluid also changes from water white transparency (A4-2 group) from coffee-like (A1-2 group), yellow (A2-2 group), light yellow (A3-2 group).
Continue and above-mentioned each group of Antrodia camphorata dilution (A1-1 ~ A4-1 group) and each group Antrodia camphorata filtered fluid (A1-2 ~ A4-2 group) are analyzed each group of particle size with a particle size analyzer.Please refer to shown in Fig. 3, the particle diameter of A1-1 group Antrodia camphorata dilution up to 6,000nm, the particle diameter of the A1-2 group Antrodia camphorata filtered fluid filtered by 0.22 μm of filtering membrane still up to 4,000nm; And by after 1,000 × dilution, the particle diameter of Antrodia camphorata dilution (A3-1 group) and Antrodia camphorata filtered fluid (A3-2 group) drops to about 2,000nm and 500nm respectively; And, dilution 10, Antrodia camphorata dilution (A4-1 group) particle diameter after 000 × dilution is 258nm, the particle diameter of the A4-2 group Antrodia camphorata filtered fluid filtered by 0.22 μm of filtering membrane has then been less than the detection limit of this particle size analyzer, be shown in this A4-2 group Antrodia camphorata filtered fluid, its filter footpath particle is obviously removed, and the particulate matter of Antrodia camphorata extract can be avoided the impact of subsequent analysis result.
In addition, continue and each group of Antrodia camphorata dilution (A1-1 ~ A4-1 group) is analyzed with three-dimensional fluorescence detection system, its result respectively corresponding order shows as shown in Fig. 4 A ~ 4D, when extension rate is larger, crest location obviously moves toward low excitation/emission position, and, dilution 10,000 × the excitation/emission crest location of Antrodia camphorata dilution (A4-1 group) and fluorescence intensity level be respectively 290/380nm and 2,265(is as shown in Figure 4 D).
And, the corresponding order display of result that each group of Antrodia camphorata filtered fluid (A1-2 ~ A4-2 group) carries out analyzing with three-dimensional fluorescence detection system is as shown in Fig. 5 A ~ 5D, dilution 10,000 × the excitation/emission crest location of Antrodia camphorata filtered fluid (A4-2 group) and fluorescence intensity level be respectively 270/320nm and 3,128(as shown in Figure 5 D), Antrodia camphorata extract be diluted and be filtered to display goodly should, to promote the accuracy of analysis result before analyzing with this three-dimensional fluorescence detection system.
Please refer to shown in Fig. 6, is the second preferred embodiment of the present invention, and compared to the first preferred embodiment, this second preferred embodiment is before this three-dimensional fluorescence detection system is analyzed, and mushroom Mushrooms sample is carried out separation and purification through a part screening layer analysis system.
In details of the words, better photodiode array detector of can arranging in pairs or groups (the diode array detector of molecular screening tomographic system, be called for short DAD) carry out the mensuration of organic substance signal, to carry out the absorption scanning of multistage wavelength, and, separation and purification goes out the mushroom Mushrooms sample that molecular weight is a purifying of 100 ~ 10000Da, the continuous mushroom Mushrooms sample analyzing this purifying with this three-dimensional fluorescence detection system.
For the mushroom Mushrooms analytical approach of proved second preferred embodiment also can effective constituent contained by effectively qualitative and quantitative mushroom Mushrooms sample, then get above-mentioned A4-2 group Antrodia camphorata filtered fluid and test.
In this second preferred embodiment, molecular screening tomographic system can select a liquid chromatography system (L-7100, purchased from Hitachi, Japan), and diode array detector of arranging in pairs or groups carries out the mensuration of organism signal.Preferably, the chromatography tubing string of this liquid chromatography system is chosen as TSK HW-55S(purchased from Tosoh, USA), its internal diameter and length are respectively 7.8mm and 300mm, and the inside stuffing of this chromatography tubing string is hydroxylation metering system based polyalcohol (hydroxylated methacrylicpolymer), particle diameter and mean pore size are respectively 20 ~ 40 μm and 125
.The flow velocity of this molecular screening tomographic system is 0.5mL/min, and movement is that phosphate buffer is (with 2.4mMNaH mutually
2pO
4, 1.6mM Na
2hPO
4and 25mM Na
2sO
4the buffer solution be hybridly prepared into, pH is 6.8, and ionic strength is 100mM).
And, this second preferred embodiment is respectively 410 with molecular weight, 000,150,000,50,000,25,000, the molecular weight standards of 5,000 and 1,000Da, and acetone (molecular weight is 58) draws the rating curve of hold-up time and molecular size range, its result as shown in Figure 7, and calculating a regression curve according to this rating curve is Log (M)=8.336-0.263 × Ve(R=0.997).The below continuous corresponding molecular weight calculating fruiting Mushrooms sample effective constituent with this regression curve of test.
Please refer to shown in Fig. 8, above-mentioned A4-2 group Antrodia camphorata filtered fluid is after analyzing with molecular screening tomographic system, and its flowing lotion is 19 ~ 25 minutes in the hold-up time, and molecular weight ranges falls within 102 ~ 104Da occurs obvious crest.
And, continuing will allow flowing lotion enter three-dimensional fluorescence detection system with liquid phase flow system (flow cell), and the organism analyzed in place appearance in 23 minutes, result as shown in Figure 9, have the two obvious crests in place, correspondence position (exciting crest/transmitting crest) and intensity are 230/320nm(628.4) and 290/320nm(490.9).
Result is obtained by confirming to analyze with this second preferred embodiment, for Antrodia camphorata index components 4,7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene (4,7-dimethoxy-5-methyl-1,3-benzodioxole) and horse together with No. 1 (dehydrosulphurenic acid, belong to lanostane steroid compound), this test continues to be tested with these two kinds of Antrodia camphorata index components (analytical concentration is for 20ppm).
Please refer to shown in Figure 10 A and Figure 10 B, be respectively Antrodia camphorata index components 4,7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene and horse together with No. 1 with molecular screening tomographic system analysis result, wherein, there is obvious UV absorption value in 20 ~ 25 minutes in its flowing lotion, its molecular weight is about 102 ~ 103Da, and, Antrodia camphorata index components 4,7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene has obvious absorption value in 200 and 380nm, and horse then has obvious absorption value in 270,310 and 380nm respectively together with No. 1.Therefore although the molecular weight of these two kinds of Antrodia camphorata index components is close, still difference identification can be carried out by different absorbing wavelength.Further, with results contrast shown in Fig. 8, display Antrodia camphorata filtered fluid comprises this two kinds of Antrodia camphorata index components.
And, flowing lotion is analyzed with three-dimensional fluorescence detection system, be 23min in the hold-up time, its corresponding molecular size range is 100 ~ 200Da, wherein, 4,7-bi-methoxy-5-methyl isophthalic acid, the crest location (exciting crest/transmitting crest) of 3-dioxygen benzene and intensity are 220/320nm(243.7) and 270/330nm(104.5) (result as shown in Figure 11 A), further, horse is 230/330nm(285.3 together with the crest location (exciting crest/transmitting crest) of No. 1 and intensity) and 280/330nm(225.6) (result as shown in Figure 11 B).Further, with results contrast shown in Fig. 9, what show Antrodia camphorata filtered fluid comprises this two kinds of Antrodia camphorata index components.
Moreover, the result that two kinds of Antrodia camphorata index components are analyzed with molecular screening tomographic system, the main hold-up time of these two kinds of Antrodia camphorata index components is 23 minutes, and jointly there is crest in determined wavelength 205 and 380nm two place, therefore, the calibration curve of variable concentrations is drawn out for this two place, result please refer to shown in Figure 12 A ~ Figure 12 D, wherein, 4, 7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene (as illustrated in fig. 12) and horse together with No. one (as shown in Figure 12 B) under the concentration between 1 ~ 25mg/L, in 205 and absorption value corresponding to 380nm, there is better linear relationship with UV, thus contained by A4-2 group Antrodia camphorata dilution 4 of above-mentioned Fig. 9 record can be pushed back accordingly, 7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene and horse are together with a content, its result shows, 4, 7-bi-methoxy-5-methyl isophthalic acid, 3-dioxygen benzene in the concentration of 205 and 380nm position species be 31, 514 and 44, 802ppm, and horse is 49 together with No. one in 205 and 380nm concentration, 747 and 45, 187ppm.
And, when horse reaches more than 10ppm together with the concentration of No. one, separately there is crest in wavelength 270nm place, also drafting rating curve is carried out for this crest, its result as shown in figure 13, and can push back the concentration of 4-2 group Antrodia camphorata filtered fluid that above-mentioned Fig. 9 records accordingly and should be 170,500mg/L.
In addition, separately whether can be used for the mushroom Mushrooms analytical approach of this second preferred embodiment of Cordyceps sinensis index components cordycepin (3-deoxyadenosine) test the mushroom Mushrooms sample analyzing non-Antrodia camphorata, this test is tested with a Cordyceps sinensis index components (concentration is the cordycepin of 10ppm).
With molecular screening tomographic system analysis result as shown in figure 14, and the continuous result analyzed through three-dimensional fluorescence detection system by flowing lotion as shown in figure 15 for Cordyceps sinensis index components.Can be learnt by this test findings, this Cordyceps sinensis index components has obvious crest respectively at 290/320nm and 230/330nm two place, and its intensity is respectively 384.1 and 335.5; Accordingly, this second preferred embodiment also can be used for and analyzes Cordyceps sinensis index components.
Comprehensively above-mentioned, mushroom Mushrooms analytical approach of the present invention is the molecular characterization by detecting mushroom Mushrooms extract, can effective constituent that is qualitative and quantitatively this mushroom Mushrooms extract, to reach the effect promoting commercially available mushroom Mushrooms extract quality.
And, mushroom Mushrooms analytical approach of the present invention is the excitation wavelength and emission wavelength that have by the molecular structure of mushroom Mushrooms extract, to calculate the active constituent content of this mushroom Mushrooms extract, significantly can reduce the time detected and analyze, reach effect of quick testing sample screening.
Just above-described content, is only preferred embodiment of the present invention, when not limiting the scope of the present invention with this; Therefore all simple equivalences done according to the present patent application the scope of the claims and creation description change and modify, and all should still remain within the scope of the patent.
Claims (4)
1. a mushroom Mushrooms analytical approach, is characterized in that, comprises:
Step (a): mushroom Mushrooms sample is provided;
Step (b): analyze this mushroom Mushrooms sample with three-dimensional fluorescence detection system, the excitation wavelength of this three-dimensional fluorescence detection system and emission wavelength are respectively 250 nm and 310 nm.
2. mushroom Mushrooms analytical approach as claimed in claim 1, it is characterized in that, before this step (b), this mushroom Mushrooms sample of step (a) is isolated the mushroom Mushrooms sample of purifying through molecular screening tomographic system, the continuous mushroom Mushrooms sample analyzing this purifying with this three-dimensional fluorescence detection system.
3. mushroom Mushrooms analytical approach as claimed in claim 2, it is characterized in that, the molecular weight of the mushroom Mushrooms sample of this purifying is 100 ~ 10000 Da.
4. the mushroom Mushrooms analytical approach as described in claim 1,2 or 3, is characterized in that, this mushroom Mushrooms sample in this step (a) be mushroom Mushrooms extract through being diluted to clear, and carry out filtration with 0.22 μm of filtering membrane and obtained.
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Application publication date: 20150318 |
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